Koon Ho Wong’s lab, University of Macau
Load feature file and data (bw files stored as GRanges object).
# bw files data
data_files <- system.file("extdata/sysdata.rda" , package = "FungalSporeAnalysis")
load(data_files)
# genome feature file
sql_lite_dir <- system.file("extdata","sqllite" , package = "FungalSporeAnalysis")
sql_files <- list.files(path = sql_lite_dir , full.names = T)
names(sql_files) <- basename(sql_files)
# gene lists with expression
gene_list_dir <- system.file("extdata","genesets" , package = "FungalSporeAnalysis")
gene_list_files <- list.files(path = gene_list_dir , full.names = T)
names(gene_list_files) <- basename(gene_list_files)PIC profiles ie. RNAP-II, TBP and TFII-B in spores of A. nidulans generated using genelist_specific_profileplot function.
gene_list <- readr::read_delim(gene_list_files["an_spore_pol2.txt" ], delim="\t", col_names = FALSE)
feature_txDb <- AnnotationDbi::loadDb(sql_files["an_feature_file_s10_m04_r07.sqlite"])
# RNAP-II
genelist_specific_profileplot(feature_txDb =feature_txDb,bw_files = "pol2_veA_wt_spore", genelist=gene_list, output_name="An_Spore_Pol2", ymin=3,max_key = 10, min_key = 0, ymax = 5.5, palette = "white_red")
# TBP
genelist_specific_profileplot(feature_txDb =feature_txDb,bw_files = "veA_wt_TBP_spore", genelist=gene_list,max_key=4.5,min_key = 0, output_name="An_Spore_TBP", ymin=3, palette = "white_green", ymax = 5.5)
# TFII-B
genelist_specific_profileplot(feature_txDb =feature_txDb,bw_files = "TFIIB_veA_wt_spore", genelist=gene_list,max_key=5,min_key = 1, output_name="An_Spore_TFIIB", ymin=3, palette = "white_blue", ymax = 5.5)
Distribution of actively transcrbing genes ranked by RNAP-II values in data RNAP-II and RNASeq data.
genelist <- readr::read_delim(gene_list_files["an_spore_pol2_for_percentilelineplot.txt"],delim="\t", col_names=FALSE)
# RNAP-II
lineplot_for_bw(feature_txDb, genelist =genelist,bw_file="pol2_veA_wt_spore", output_name = "pol2_veA_wt_spore")
# mRNA levels
lineplot_for_bw(feature_txDb, genelist=genelist, bw_file="An_d3_spore_mRNA_hisat2", output_name = "An_d3_spore_mRNA_hisat2", tss=FALSE)Distribution of actively transcrbing genes ranked by RNAP-II values and control genes(non-transcribing genes) in H3 data.
# Histone H3 occupancy
genelist <- readr::read_delim(gene_list_files["an_spore_pol2_for_H3percentile.txt"],delim="\t", col_names=FALSE)
lineplot_for_bw(feature_txDb, genelist =genelist,bw_file="H3_an_spore", output_name = "H3_an_spore", expression_value = FALSE)
Different histone mark’s occupancy at actively transcribing genes in spore. Here, histone mark signals are normalized to H3. Plots are generated using profiles_normalized_by_control function. First, load RNAP-II signals to rank the histone marks and a genelist of control genes which are lowly transcribing or non-transcribing genes. And then generate normalized profiles of H3Ac, H3K4me3 and H3K36me3 marks.
genelist_1 <- readr::read_delim(gene_list_files["an_spore_pol2.txt"], delim="\t", col_names = FALSE)
genelist_2 <- readr::read_delim(gene_list_files["an_spore_pol2_controlgenes.txt"], delim="\t", col_names = FALSE)
# H3Ac occupancy
profiles_normalized_by_control(feature_txDb = feature_txDb,bw_test = "H3AC_veA_wt_spore", bw_control = "H3_an_spore",genelist_1 = genelist_1,genelist_2 = genelist_2, ymax = 3.8,ymin=0.5, output_name = "H3Ac_veA_wt_spore")
# H3K4me3
profiles_normalized_by_control(feature_txDb = feature_txDb,bw_test = "H3K4me3_veA_wt_spore", bw_control = "H3_an_spore",genelist_1 = genelist_1,genelist_2 = genelist_2, ymax = 6,ymin=0.3, output_name = "H3K4me3_veA_wt_spore")
# H3K36me3
profiles_normalized_by_control(feature_txDb = feature_txDb,bw_test = "H3K36me3_veA_wt_spore", bw_control = "H3_an_spore",genelist_1 = genelist_1,genelist_2 = genelist_2, ymax = 2,ymin=0.5, output_name = "H3K36me3_veA_wt_spore")
# tRNA feature file
feature_txDb <- AnnotationDbi::loadDb(sql_lite_file["an_feature_file_s10_m04_r07_tRNA.sqlite"])
# tRNA signal file
gene_list <- readr::read_delim(gene_list_files["an_spore_tRNA.txt"], delim="\t", col_names = FALSE)
# TFIII-B
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "AN3116_HA_spore",ymax = 280, ymin = 10, max_key = 59, min_key = 0, output_name = "AN3116_HA_spore", log2 = FALSE, palette = "cream_green")
# TBP
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "veA_wt_TBP_spore",ymax = 46, ymin = 8, max_key = 59, min_key = 0, output_name = "TBP_veA_wt_spore_tRNA", log2 = FALSE, palette = "cream_green")
# TFIII-C
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "AN7997_HA_spore",ymax = 26, ymin = 8, max_key = 59, min_key = 0, output_name = "AN7997_HA_spore", log2 = FALSE, palette = "cream_green")
# Rpo31
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "AN10316_HA_spore",ymax = 270, ymin = 10, max_key = 150, min_key = 0, output_name = "AN10316_HA_spore", log2 = FALSE, palette = "cream_brown")
# Rpc40
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "AN2415_HA_spore",ymax = 180, ymin = 10, max_key = 150, min_key = 0, output_name = "AN2415_HA_spore", log2 = FALSE, palette = "cream_brown")
# Rpc11
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = "AN4219_myc_spore",ymax = 225, ymin = 10, max_key = 150, min_key = 0, output_name = "AN4219_myc_spore", log2 = FALSE, palette = "cream_brown")
# rRNA feature file
feature_txDb <- AnnotationDbi::loadDb(sql_files["aniger_feature_file_s01_m07_r09_5SrDNA.sqlite"])
# rRNA signal file
gene_list <- readr::read_delim(gene_list_files["an_spore_5SrDNA.txt"], delim="\t", col_names = FALSE)
# Initiation factors
bw_files <- c("AN3116_HA_spore_niger","veA_wt_TBP_spore_niger", "AN7997_HA_spore_niger")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = bw_files,ymax = 2200, ymin = 0, max_key = 1050, min_key = 0, output_name = "an_spore_initiationfactor_5SrDNA", log2 = FALSE, palette = "cream_green", rename = TRUE)
# RNAP-III subunits
bw_files=c("AN10316_HA_spore_niger","AN2415_HA_spore_niger", "AN4219_myc_spore_niger")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = bw_files,ymax = 2500, ymin = 0, max_key = 1050, min_key = 0, output_name = "an_spore_pol3_5SrDNA", log2 = FALSE, palette = "cream_brown", rename = TRUE)
# A. nidulans
feature_txDb <- AnnotationDbi::loadDb(sql_files["an_feature_file_s10_m04_r07.sqlite"])
bw_files <- c("pol2_veA_wt_spore","input_an_3dspore_polII")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = NULL, bw_files = bw_files,top_line = FALSE, max_key = 59, min_key = 0, output_name = "an_pol2_input", log2 = FALSE, palette = "cream_brown", rename = TRUE)
# A. fumigatus
feature_txDb <- AnnotationDbi::loadDb(sql_files["af_feature_file_s03_m05_r07.sqlite"])
bw_files <- c("Af293_spore_2d_polii","input_af_3dspore_polII")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = NULL, bw_files = bw_files,top_line = FALSE, max_key = 30, min_key = 0, output_name = "af_pol2_input", log2 = FALSE, palette = "cream_green", rename = TRUE)
# P. marneffei
feature_txDb <- AnnotationDbi::loadDb(sql_files["pm_feature_file_fungiDb41.sqlite"])
bw_files <- c("pm_3day_spore_pol2","input_pm_21dspore_polII")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = NULL, bw_files = bw_files,top_line = FALSE, max_key = 30, min_key = 0, output_name = "pm_pol2_input", log2 = FALSE, palette = "cream_pink", rename = TRUE)
dat <- readr::read_delim(gene_list_files["an_spore_hypha_specificgenes.txt"], delim="\t", col_names = TRUE)
input_data <- dat %>% dplyr::filter(class=="spore_maturation")
ggplot_heatmap(input_data ,threshold = TRUE, output_name = "An_spores_maturation_genes_exprsn")
GO enrichment of actively transcribing genes of A. nidulans, A. fumigatus and P. marneffei.
data <- readr::read_delim(gene_list_files["an_af_pm_spores_GO.txt"], col_names = TRUE, delim="\t")
GO_diamondplot(data, output_name = "An_Af_Pm_GO", palette="three_color")GO enrichment of actievly transcribing genes in A. nidulans spores under temperature, salt and Zn- stress.
data <- readr::read_delim(gene_list_files["an_spore_stress_GO.txt"], col_names = TRUE, delim="\t")
data_subset <- data %>% dplyr::filter(class=="4C"|class=="42C")
GO_diamondplot(data=data_subset, output_name = "An_spore_temperature", palette="two_color")
GO enrichment of actievly transcribing genes in A. fumigatus spores under salt and Zn- stress.
data <- readr::read_delim(gene_list_files["af_spore_stress_GO.txt"], col_names = TRUE, delim="\t")
data_subset <- data %>% dplyr::filter(class=="Zn")
GO_diamondplot(data=data_subset, output_name = "Af_spore_Zn", palette="two_color")
feature_txDb <- AnnotationDbi::loadDb(sql_files["an_feature_file_s10_m04_r07.sqlite"])
bw_files <- c("pol2_veA_wt_spore","6day_pol2_spore","17day_pol2_spore")
gene_list <- readr::read_delim(gene_list_files["an_spore_pol2.txt"], delim="\t", col_names = FALSE)
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = bw_files,ymax = 62, ymin = 10, max_key = 60, min_key = 0, output_name = "an_pol2_3d_6d_17d", log2 = FALSE, palette = "cream_brown", rename = TRUE)feature_txDb <- AnnotationDbi::loadDb(sql_files["af_feature_file_s03_m05_r07.sqlite"])
gene_list <- readr::read_delim(gene_list_files["af_spore_pol2.txt"], delim="\t", col_names = FALSE)
# plot1: 3-day, plot2: 17-day
bw_files <- c("Af293_spore_2d_polii", "af293_spore_17d_polii")
genelist_specific_profileplot(feature_txDb = feature_txDb, genelist = gene_list, bw_files = bw_files,ymax=30,ymin=10, max_key = 30, min_key = 0, output_name = "af_3day_17day_spore_pol2", log2 = FALSE, palette = "cream_green", rename = TRUE)RNAP-II and RNASeq correlation was computed using normalised read count over the gene-body. Scatterplot of normalised read count and FPKM values were plotted using scatter plot functionality of FungiExpresZ v0.0.4
For histone marks and TF’s, normalised bw files are compared using multiBigwigSummary function from deeptools.
multiBigwigSummary bins -bs 1000 -b an_spore_H3_AGAACACC_CL1019Mix_normalized.bw an_spore_H3_mix35_TCCAGCCTCT_normalized.bw -outRawCounts an_histone_spore_count_mat.tab --chromosomesToSkip mito_A_nidulans_FGSC_A4 -l an_spore_H3_set1 an_spore_H3_set2dat <- read.delim("inst/extdata/correlation_data/an_histone_spore_count_mat.tab",sep="\t", header = TRUE)
gp <- bw_corr(dat, pattern = ".*_spore_(.*)_(.*)") 4. TF’s
dat <- read.delim("inst/extdata/correlation_data/an_tbp_tf2b_spore_count_mat.tab",sep="\t", header = TRUE)
gp <- bw_corr(dat, pattern = ".*_spore_(.*)_(.*)")